Inhibition of Pseudomonas aeruginosa Biofilm Formation by ...downloads.hindawi.com/journals/bmri/2017/9584703.pdf · ResearchArticle Inhibition of Pseudomonas aeruginosa Biofilm Formation

Research ArticleInhibition of Pseudomonas aeruginosa Biofilm Formationby Traditional Chinese Medicinal Herb Herba patriniae

Bo Fu1 QiaolianWu1 Minyan Dang1 Dangdang Bai1 Qiao Guo1

Lixin Shen1 and Kangmin Duan12

1Key Laboratory of Resources Biology and Biotechnology in Western China Ministry of Education College of Life SciencesNorthwest University Xirsquoan Shaanxi 710069 China2Department of Oral Biology amp Medical Microbiology Rady Faculty of Health Sciences University of Manitoba 780 Bannatyne AveWinnipeg MB Canada R3E 0W2

Correspondence should be addressed to Lixin Shen shenlxnwueducn and Kangmin Duan kangminduanumanitobaca

Received 16 August 2016 Accepted 5 January 2017 Published 9 March 2017

Academic Editor P Nithyanand

Copyright copy 2017 Bo Fu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

New antimicrobial agents are urgently needed to treat infections caused by drug-resistant pathogens and by pathogens capableof persisting in biofilms The aim of this study was to identify traditional Chinese herbs that could inhibit biofilm formationof Pseudomonas aeruginosa an important human pathogen that causes serious and difficult-to-treat infections in humans AluxCDABE-based reporter system was constructed to monitor the expression of six key biofilm-associated genes in P aeruginosaThe reporters were used to screen a library of 36 herb extracts for inhibitory properties against these genes The results obtainedindicated that the extract of Herba patriniae displayed significant inhibitory effect on almost all of these biofilm-associated genesQuantitative analysis showed thatH patriniae extract was able to significantly reduce the biofilm formation and dramatically alteredthe structure of the mature biofilms of P aeruginosa Further studies showed H patriniae extract decreased exopolysaccharideproduction by P aeruginosa and promoted its swarming motility two features disparately associated with biofilm formationTheseresults provided a potential mechanism for the use ofH patriniae to treat bacterial infections by traditional Chinese medicines andrevealed a promising candidate for exploration of new drugs against P aeruginosa biofilm-associated infections

1 Introduction

Pseudomonas aeruginosa is a remarkably adaptive bacterialpathogen which can cause persistent infections in burnpatients immune-compromised patients and individualswith the genetic disease cystic fibrosis It is one of the mostprevalent nosocomial pathogens and the infections causedby P aeruginosa can be very serious and life-threatening [1]

Biofilm formation is a major characteristic of P aerugi-nosa chronic infections [2 3] P aeruginosa cells in biofilmsare surrounded by exopolysaccharides and form a struc-tured aggregates and these cells exhibit increased resistanceto antibiotics and other adversary agents [3ndash6] Infectionscaused by biofilm-forming P aeruginosa such as those incystic fibrosis lung are almost impossible to eradicate [7]There is an urgent need to find novel antimicrobial agents tocontrol such infections [8 9]

Traditional Chinese medicines (TCMs) have been widelyused to treat infectious diseases for more than a thousandyears in ChinaMany components constituents of TCMs havebeen found to be very effective in treating bacterial infec-tions such as gastritis stomatitis dermatitis and bacterialpneumonia [10] However the mechanisms of these herbs intreatment infectious diseases are mostly unknown

Herba patriniae is a perennial herbal of TCM whichcontains various beneficial ingredients such as amino acidsvitamins minerals alkaloids tannins and saponins It hasbeen reported to have functions of antioxidant antibacterialantiviral blood-activating and stasis-eliminating promotingregeneration of liver cells and anxiety-alleviatingTheboilingwater extracts of H patriniae had been identified havinganticyanobacteria activity againstMicrocystis aeruginosa [11]

In this study 36 extracts of 18 Chinese herbs that arecommonly used for treating infection-like symptoms were

HindawiBioMed Research InternationalVolume 2017 Article ID 9584703 10 pageshttpsdoiorg10115520179584703

2 BioMed Research International

Table 1 Bacterial strains used in this study

Strainsplasmids Description Source

E coli DH10B F-mcrA(mrr-hsdRMS-mcrBC)80dlacZ ΔM15ΔlacX74 deoRrecA1 endA1 araD139 Δ(ara leu)7697 galU galK120582-rpsl nupG Invitrogen

P aeruginosa PAO1 Wild type This lab

pMS402 Expression reporter plasmid carrying the promoterlessluxCDABE Kanr Tmpr This lab

pKD-pslM pMS402 containing pslM promoter region Kanr Tmpr This studypKD-pelA pMS402 containing pelA promoter region Kanr Tmpr This studypKD-algU pMS402 containing algU promoter region Kanr Tmpr This studypKD-ppyR pMS402 containing ppyR promoter region Kanr Tmpr This studypKD-algA pMS402 containing algA promoter region Kanr Tmpr This studypKD-bdlA pMS402 containing bdlA promoter region Kanr Tmpr This study

Table 2 Reporter genes and primer sequences used

PAnumber Gene Function Primer Sequence (51015840 rarr 31015840)

PA0762 algU RNA polymerase sigma factor pKD-algU-S GCACTCGAGAGGATGCCTGAAGACCTCpKD-algU-A GTAGGATCCGATGGCGATCCGATACAG

PA2243 pslM Succinate dehydrogenasefumarate reductase flavoprotein

pKD-pslM-S ATCCTCGAGCGGTGCGCAAGAAGACCpKD-pslM-A GTTGGATCCCGTAACGCTCGCCCAGTT

PA3064 pelA Glycoside hydrolasedeacetylase

pKD-pelA-S CGTCTCGAGCTTTCCACTTTGCCACAGpKD-pelA-A TACGGATCCTACCAGAACGCCACGCT

PA3551 algAPhosphomannose isomeraseguanosine 51015840-diphospho-D-mannosepyrophosphorylase

pKD-algA-S TGACTCGAGTGAAGGCGGACTGAGACpKD-algA-A TATGGATCCGCGACCAGTTTCATC

PA2663 ppyR psl and pyoverdine operon regulator pKD-ppyR-S GCACTCGAGCACTTCTTCTGCTACAGCpKD-ppyR-A GTAGGATCCAGCACTTGCACAGCAGAC

PA1423 bdlA Biofilm dispersion locus A pKD-bdlA-S GCACTCGAGCGTCATATTTCCGACGAApKD-bdlA-A TCAGGATCCGTAGTCTTCCGATTGCG

screened for inhibitory effect against P aeruginosa biofilmsWe found that the extract of the Chinese herb H patriniaesignificantly inhibited the expression of the genes associatedwith biofilm formation in P aeruginosa PAO1 It reducedexopolysaccharide production and biofilm formation andthen altered the structure of the mature biofilms

2 Materials and Methods

21 Bacterial Strains andCulture Conditions Bacterial strainsused in this study are listed in Table 1 All the strains werecultured in LB (Luria Bertani) broth at 37∘C with orbitalshaking at 200 rpm or on LB agar plates supplemented withantibiotic of kanamycin (Kan 50 120583gmL) or trimethoprim(Tmp 300 120583gmL) where appropriate

22 Traditional Chinese Medicine Extraction TraditionalChinese medicinal herbs were selected according to theirefficacy in treatment of infection-like symptoms in Chinesemedicine They were obtained from local pharmacy (Yikang

Pharm chain store China) The pulverized herb was firstlyimmerged in 75 ethanol or deionized water respectivelyfor 2 h and then boiled for 2 h additionally (weight to solventvolume was 1 5) The extracts were filtered by filter paperand were evaporated under vacuum at 35∘C using a rotaryevaporator (Buchi Switzerland) The concentrated extractswere then freeze-dried using a lyophilizer and stored atminus80∘CThewater extractswere redissolved in deionizedwaterand ethanol extracts were redissolved in methanol and thensterilized immediately with 022120583m Iwaki filter before use

23 Gene ExpressionAssay Key genes algU [12] pslM [13 14]pelA [15] algA [16] ppyR [17] and bdlA [18] that are knownto be involved in biofilm formation in P aeruginosa wereselected to construct luxCDABE-based promoter-reporterfusions (Table 2) The reporters were constructed as previ-ously described [19 20] and were subsequently transformedinto PAO1 Using luxCDABE-based reporters gene expres-sion in liquid cultures was measured by light luminescence(in counts per second) in a Victor3 multilabel plate reader

BioMed Research International 3

(Perkin-Elmer) Strains containing different reporters werecultivated overnight in LB broth supplemented with Tmp(300 120583gmL) and then diluted to an optical density at 600 nmof 02 The diluted cultures were used as inoculants Afteradditional 2 h incubation 5 120583L cultures were inoculated intoparallel wells on a 96-well black plate containing 93 120583Lmedium and 2120583L herbal extract in different concentrations50 120583L mineral oil was added to the wells to prevent evapora-tion Promoter activities weremeasured every 30min for 24 hin a Victor multilabel plate reader and bacterial growth wasmonitored by measuring OD

600at the same time

24 Gene Expression Measurement by Real-Time Quantita-tive PCR (RT-qPCR) Bacterial RNA was extracted usingRNAprep Pure CellBacteria Kit (TIANGEN) 1 120583g of RNAsample was reverse transcribed to cDNA using 1st StrandcDNA Synthesis Kit (Takara) Real-time qPCR was perfor-med using SuperReal PerMix Plus (SYBR Greenprobe)(TIANGEN) and primers specific for algU (Forward 51015840-AACACCGCGAAGAACCACCT-31015840 Reverse 51015840-ATCTCA-TCCCGCAACATCGCG-31015840) algA (Forward 51015840-AGAACT-GAAGAAGCACGACG-31015840 Reverse 51015840-TTCTCCATCACC-GCGTAGT-31015840) pelA (Forward 51015840-ATGGCTGAAGGTATG-GCTG-31015840 Reverse 51015840-AGGTGCTGGAGGACTTCATC-31015840)and pslM (Forward 51015840-CTATGACGCACGGCAACTGG-31015840reverse 51015840-CGCCATTGACCAGGTGCAT-31015840) The valuesobtained were normalized to the housekeeping gene proC(Forward 51015840-CAGGCCGGGCAGTTGCTGTC-31015840 reverse51015840-GGTCAGGCGCGAGGCTGTCT -31015840)

25 Quantification of the Biofilm Formation Biofilm forma-tion was measured in 96-well polystyrene microtiter plateas described previously with minor modifications [21] 10 120583Lof overnight cultures of PAO1 was added to 90 120583L fresh LBbroth containing 160 120583g H patriniae water extract Samevolume of deionized water was used as control After beingcultured with shaking at 200 rpm for 1 h the plates were keptat stationary state at 37∘C for 3 days and 7 days with amediumreplacement at every 24 h interval Then wells were washedtwice with deionized water gently to remove the planktoniccells The sedentary cells were stained with 1 (vv) crystalviolet solution for 15min Unbound dye in the wells werewashed off by deionized water before 200120583L of 95 ethanolwas added to dissolve the crystal violet stainsThe absorbanceof the solutions was then measured at 570 nm [7 22]

26 Biofilm Imaging by Silver Staining Method To exam-ine the biofilm structure silver staining method was usedas described previously with some modifications [23 24]Similar to the above described biofilm assay biofilms weregrown on cover slip (8mm diameter) placed at bottom ofthe wells of 24-well plates 200120583L of fresh bacterial culturewas inoculated to 18mL TSB supplemented with 32mgH patriniae The plates were incubated at 37∘C with TSBmedium replaced every 24 h After 3 days or 7 days ofcultivation the cover slip was taken out and washed threetimes with saline water to remove the planktonic cells Thecover slip was immersed in 25 glutaraldehyde for 1 h for thecells fixation and rinsed with distilled water for 1min Then

the cover slip was immersed in saturated calcium chloridesolution for 15min and rinsed with distilled water for 5minThe biofilms on the cover slip were then stained with 5silver nitrate for 15min followed by 1hydroquinone colour-rendering for 2min and then rinsed with distilled waterfor 1min Fixation was treated with 5 sodium thiosulfatefor 1min followed by a final water rinse The cover slipwas placed on an inverted optical microscope for biofilmstructure observation

27 Swarming Motility Assay Swarming assay was carriedout as previously reported [25] The medium used forswarming motility assay consists of nutrient broth (08)glucose (05) and agar (05) The plates were dried atroom temperature overnight before being used 2 120583L of Paeruginosa PAO1 culture (OD600 = 05)mixedwith 10667120583gof H patriniae water extract was spotted onto the swarmingplate and the one with deionized water was used as blankcontrol Plates were incubated at 37∘C for 24 h before theswarming diameter was measured

28 Measurement of Exopolysaccharide Production Over-night culture of PAO1 (OD600 = 0005) was spotted ontoCongo red plates (1 Tryptone 1 agar 4 Congo redand 2 Coomassie blue) with or without H patriniae waterextract The amount of H patriniae water extract was 64 120583gThe colony morphology and staining were observed after 3days of incubation at 37∘C [22]

3 Results and Discussion

31 Screening for Herbs with Inhibitory Effect on P aerugi-nosa Biofilm-Associated Genes Eighteen traditional Chinesemedicinal herbswere selected because of their commonusagefor infection-like symptoms Both boiling water extractsand ethanol extracts were obtained and used to screen forantibiofilm activities Since P aeruginosa biofilm formation isdirectly associated with the activity of several known geneswe constructed luxCDABE-based reporters to examine theeffect of herb extracts on these genes The effects of thecrude extracts on the expression of these biofilm-associatedgenes (algU pslM pelA algA ppyR and bdlA) are presentedin Table 3 The results indicate that different herbal extractsexhibited various degrees of inhibitory effects on these genesThe water extract ofH patriniae showed the most significanteffect on the expression of algU algA pslM and bdlAExamples of the gene expression profiles in the presence ofH patriniae are shown in Figure 1

To confirm the results obtained from the lux-basedreporter assay real-time qPCRwas carried out using bacterialRNA samples isolated in the presence and absence of HpatriniaeThe results are shown in Table 4 In agreement withthe results from the reporter assay the mRNA levels of algUalgA pslM and bdlA in PAO1 were all significantly decreasedin the presence of H patriniae extract (at 16mgmL) com-pared with those in the absence of H patriniae extract Itis noted that the inhibition of algU and algA was morepronounced in the qPCR assay


Table3Eff

ecto

fwater

andethano

licherb

extractson

biofi

lm-associatedgenese

xpression

TheE

nglishnameo

fherbs

Water

extract

TheE

nglishnameo

fherbs

Ethano

licextract

algU

pslM

pelA

algA

ppyR

bdlA

algU

pslM

pelA

algA

ppyR

bdlA

Atractylodeslancea

+15

0minus25

00

0Atractylodeslancea

00

00

00

Cortex

fraxini

0minus3

minus15

minus1

00

Cortex

fraxini

minus18

minus25minus05minus15

00

Cyrtom

ium

fortun

ei0

minus15

minus2

minus1

00

Cyrtom

ium

fortun

ei0

00

00

0Erodium

stephanianu

mWilld

aa

aa

aa

Erodium

stephanianu

mwilld

aa

aa

aa

Foliu

martemisiae

Argyi

minus2

0minus2

0minus15

0Foliu

martemisiae

argyi

00

minus2

0minus15

0Fructusq

uisqua

lisa

aa

aa

aFructusq

uisqua

lisa

aa

aa

aHerba

agrim

oniae

aa

aa

aa

Herba

agrim

oniae

aa

aa

aa

Herba

patriniae

minus2


0minus2

Herba

patriniae

00

00

minus15

minus1

Herba

scutellariaeb

arbatae

00

0minus1

00

Herba

scutellariaeb

arbatae

00

00

0minus15

Pomegranaterin

dminus15

minus12

0minus1

00

Pomegranaterin

dminus15

minus1

minus1minus05

00

Portulacae

herba

+10

0+15

00

Portulacae

herba

00

00

00

Radixpaeoniae

minus05minus05

00

minus1

0Ra

dixpaeoniae

00

00

00

Radixsanguisorbae

minus15

minus1minus15

minus1minus05

0Ra

dixsanguisorbae

minus16

minus15

00

minus05

0Ra

dixlithospermi

0minus12

minus2

0minus1

0Ra

dixlithospermi

00

00

00

Scrophulariae

minus2

+15

00

00

Scrophulariae

minus05

0minus1

00

0Sm

oked

plum

aa

aa

aa

Smoked

plum

aa

aa

aa

Taraxacum

mongolicum

00

00

00

Taraxacum

mongolicum

0+2

00

00

Tripterygium

minus15

0minus25

00

0Tripterygium

00

minus2

0minus25

0Noteherbalextra

ctsw

ereused

at16

mgmL

Thevalues

show

nrepresentm

axim

alinhibitio

n(fo

ldchangesb

etweenexperim

entswith

andwith

outh

erbextract)

ldquo+rdquor

epresentsind

uctio

nof

gene

expressio

nby

herb

extracts

ldquominusrdquorepresentsinh

ibition

ofbiofi

lmgene

expressio

nby

herb

extracts

ldquo0rdquorepresentsn

oeffecto

fbiofilm

gene

expressio

nby

herb

extracts

ldquoardquorepresentsinh

ibition

oftheg

rowth

ofbacteriaN

umbers

representfoldchangesa

tthe

timep

ointsw

here

maxim

alexpressio

nwas

reachedin

thea

bsence

ofherbalextra

ct


Expression of Herba patriniae

Expression of controlGrowth of Herba patriniae

Growth of control

0

01

02

03

04

05

06

07

08

OD

600

nm

0

500

1000

1500

2000

2500

3000

3500Ex

pres

sion

(CPS

)

3 6 9 12 15 18 21 2350Time (h)

(a)



Growth of control

0

01

02

03

04

05

06

07

OD

600

nm

0

100

200

300

400

500

600

Expr

essio

n (C

PS)

3 6 9 12 15 18 21 2350Time (h)

(b)

3 6 9 12 15 18 21 2350Time (h)

0

500

1000

1500

2000

2500

3000

Expr

essio

n (C

PS)

0

01

02

03

04

05

06

07

08

09

OD

600

nm



Growth of control

(c)

3 6 9 12 15 18 21 2350Time (d)

0

01

02

03

04

05

06

07

08

OD

600

nm

0

50

100

150

200

250

300

350

400

450

Expr

essio

n (C

PS)

Expression of controlGrowth of controlExpression of Herba patriniaeGrowth of Herba patriniae

(d)

Figure 1 Expressions of algU algA pslM and bdlA in medium with water extract of Herba patriniae Expressions of algU (a) algA (b)pslM (c) and bdlA (d) in medium (total volume of 100120583L) with 160 120583g H patriniae extract and the controls were the ones without herbalextract

The results indicate that many of these herbs have aninhibitory effect on the genes associated with biofilm forma-tion in P aeruginosaThis is somewhat not surprising becausethey all have been used for treatment of chronic bacterialinfections in traditional Chinese medicine

The effect from the water extract of H patriniae wasremarkable as it inhibited five genes tested It has beenreported that the herb had antibacterial and antiviral activityHowever it was noted that the water extract did not inhibitthe growth of P aeruginosa (Figure 1) Even though conven-tional antibiotic compounds may exist inH patriniae against

other bacteria this result indicates no such component waspresent againstP aeruginosa at least not at the concentrationsused in our experiments

Lacking of bacterial killing or growth inhibition activityhowever may not be a weakness of such herbs in treatinginfectious diseases As discussed in previous reports [26ndash29]a promising new class of antipathogenic drugs that targetvirulence factors andor biofilm formation instead of killingthe pathogens hasmany advantages in clinical use First theseantipathogenic drugs theoretically are less likely to renderdrug resistance in the pathogens because they do not assert


Table 4 The transcriptional levels of selected genes in PAO1 and PAO1 treated with H patriniae

Gene Relative mRNA level determined by ΔCt calculationWithout H patriniae With H patriniae 119875 value

algU 100 0192 0014algA 100 0090 0011pslM 100 0695 0043bdlA 100 0412 0036Note the mRNA level of each gene was normalized to that of proC The values shown represent the mean of three different tests

a selective pressure on the pathogenrsquos survival Second suchtherapeutics would unlikely affect other nonpathogenic orbeneficial bacteria that is the microbiome in the host

32 Extract of H patriniae Inhibits P aeruginosa BiofilmFormation From the gene expression results the extract ofH patriniae presumably would inhibit the biofilm formationof P aeruginosa To verify such an effect P aeruginosa biofilmformation was compared in the presence and absence ofH patriniae extract As shown in Figure 2(a) significantlyless biofilm was formed in the presence of the herb extractthan that without the extract at the irreversible attachmentstage (1 d) and mature stage (3 d and 7 d) after inoculationThe result is in agreement with the gene expression datasuggesting the H patriniae extract could reduce biofilm for-mation through inhibiting the genes associated with biofilmformation

Importantly the addition of the H patriniae also dra-matically altered the structure of the biofilms (Figure 2(b))It appears that the herb extract prevented the formation ofmature biofilms only allowing P aeruginosa to form smallercell clusters These results indicate that the water extract ofH patriniae indeed was able to inhibit P aeruginosa biofilmformation

33 Water Extract of H patriniae Inhibited P aeruginosaExopolysaccharide Production The exopolysaccharide (EPS)matrix is an important component of biofilm structure[30] We compared the exopolysaccharide production inthe presence of H patriniae extract to those without Hpatriniae extract As shown in Figure 3 P aeruginosa PAO1cells producedmore EPS shown in red by Congo red stainingthan the cells with H patriniae extract This result indicatesthat H patriniae inhibited P aeruginosa exopolysaccharideproduction and hence affected biofilm formation

34 H patriniae Water Extract Promoted PAO1 Swarm-ing Motility Upon encountering a surface the surface-associated behaviors of P aeruginosa such as biofilm forma-tion and swarming are often coregulated [31] In P aerugi-nosa swarming motility is reversely correlated with biofilmformation Examination of the swarming motility of PAO1 inthe presence and absence of the water extract of H patriniaeshowed that H patriniae promoted P aeruginosa swarm-ing motility (Figure 4(a)) The diameter of PAO1 grownwith 10667 120583g H patriniae was almost 560 cm while the

control was less than 20 cm (Figure 4(b)) Considering thereverse relationship between swarming motility and biofilmformation the enhanced swarming motility by H patriniaeextract could have contributed the inhibition of biofilmformation

Taken together the extract of H patriniae clearly inhib-ited the biofilm formation ofP aeruginosa It inhibited severalkey genes algU pslM pelA algA and bdlA that are involvedin biofilm formation H patriniae reduced exopolysaccha-ride production and promoted swarming motility Increasedmotility may reduce adhesion and enable bacteria to activelyescape the biofilm matrix to become planktonic bacteria[13 31 32] As depicted in Figure 5 multiple factorspathwaysprobably have contributed to the reduction of biofilm forma-tion and the altered biofilm structure in the presence of Hpatriniae

In a time of resistance to multiple antimicrobial agentsin pathogenic bacteria being spread there is an urgent needto develop new antibacterial agents [1 32] Drugs againstinfections that involve biofilms are particularly requiredPathogens in biofilm formation are more resistant to con-ventional antibiotics and other adversary conditions such asnutritional stress Biofilms also protect bacterial cells from theactivity of host immune response [33 34]

Traditional Chinese medicines are a valuable source fornovel antibacterial agents [35ndash37]The inhibitory effect of thewater extract of H patriniae against P aeruginosa biofilmsand biofilm-related phenotypes signifies that H patriniaeis a promising candidate for treatment of infections causedby P aeruginosa biofilms It could be used in the way oftraditional Chinese medicine or it can be explored for activecompounds

4 Conclusions

Our results indicate H patriniae extract could significantlyinhibit the expression of P aeruginosa genes associated withbiofilm formation alter the structure and prevent the forma-tion of mature biofilms It also decreased exopolysaccharideproduction and promoted swarming motility These resultsprovided a potential underlying mechanism for the use of Hpatriniae to treat bacterial infections in traditional Chinesemedicine and revealed a promising candidate for explorationof new drugs against P aeruginosa biofilm-associated infec-tions


1 3 7Time (d)

ControlHerba patriniae

0

1

2

3

4

5

6

OD

570

nm

lowast

lowast

lowastlowast

(a)

3 days 7 days

PAO1

with

Her

ba p

atrin

iae

PAO1

with

out H

erba

pat

rinia

e

(b)

Figure 2 (a) Inhibition of biofilm production of PAO1 by H patriniae extract The control did not contain any herb extract ldquolowastrdquo indicatessignificant difference between the herb group and the control group (119875 lt 005) ldquolowastlowastrdquo indicates very significant difference (119875 lt 001) (b)Micrographs of biofilms formed with and withoutH patriniae Top row PAO1 silver stained biofilms in the presence of extract ofH patriniaeafter 3 days and 7 days of incubation Bottom row PAO1 silver stained biofilms without H patriniae


(a) (b)Figure 3 Photograph of exopolysaccharide production on Congo red plates (a) Without H patriniae (b) With H patriniae The colonymorphology and staining were observed after 3 days of incubation at 37∘C

(a)

Control Herba patriniae0

1

2

3

4

5

6

7

Dia

met

er le

ngth

(cm

)

lowastlowast

(b)

Figure 4 Swarming motility of PAO1 (a) PAO1 grown with H patriniae was at right the one without herb was at left (b) The swarmingdiameter of PAO1 with H patriniae the control was the one without herb ldquolowastlowastrdquo indicates significant difference (119875 lt 001)

WithHerba patriniae

SwarmWithout

Herba patriniae

Mature biofilm

darr pelAdarr algAdarr bdlA

algU darr

pslM darr

Figure 5 P aeruginosa biofilm formation and swarming motility in the influence of H patriniae


Competing Interests

The authors declare that they have no competing interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported by grants from theNSFC (31570131)NSERC (402943-2011 RGPIN) CPSF (2014M562444) andPCSIRT (IRT 15R55)

References

[1] H M Zowawi P N A Harris M J Roberts et al ldquoTheemerging threat of multidrug-resistant Gram-negative bacteriain urologyrdquoNature Reviews Urology vol 12 no 10 pp 570ndash5842015

[2] T Bjarnsholt ldquoThe role of bacterial biofilms in chronic infec-tionsrdquo APMIS Supplementum no 136 pp 1ndash51 2013

[3] M Rybtke L D Hultqvist M Givskov and T Tolker-Nielsen ldquoPseudomonas aeruginosa biofilm infections commu-nity structure antimicrobial tolerance and immune responserdquoJournal of Molecular Biology vol 427 no 23 pp 3628ndash36452015

[4] N E Head and H Yu ldquoCross-sectional analysis of clinicaland environmental isolates of Pseudomonas aeruginosa biofilmformation virulence and genome diversityrdquo Infection andImmunity vol 72 no 1 pp 133ndash144 2004

[5] T-F Mah B Pitts B Pellock G C Walker P S Stewart andG A OrsquoToole ldquoA genetic basis for Pseudomonas aeruginosabiofilm antibiotic resistancerdquo Nature vol 426 no 6964 pp306ndash310 2003

[6] M Whiteley M G Bangera R E Bumgarner et al ldquoGeneexpression in Pseudomonas aeruginosa biofilmsrdquo Nature vol413 no 6858 pp 860ndash864 2001

[7] Q Guo W Kong S Jin L Chen Y Xu and K Duan ldquoPqsR-dependent and PqsR-independent regulation of motility andbiofilm formation by PQS in Pseudomonas aeruginosa PAO1rdquoJournal of Basic Microbiology vol 54 no 7 pp 633ndash643 2014

[8] T Rasamiravaka Q Labtani P Duez and M El Jaziri ldquoTheformation of biofilms by pseudomonas aeruginosa a review ofthe natural and synthetic compounds interfering with controlmechanismsrdquo BioMed Research International vol 2015 ArticleID 759348 17 pages 2015

[9] G H Talbot J Bradley J E Edwards Jr D Gilbert M Scheidand J G Bartlett ldquoBad bugs need drugs an update on thedevelopment pipeline from the antimicrobial availability taskforce of the infectious diseases society of Americardquo ClinicalInfectious Diseases vol 42 no 5 pp 657ndash668 2006

[10] Q Guo S Jin J Xiao D Zhang L Shen and K DuanldquoInhibition of quorum sensing-associated virulence factors inPseudomonas aeruginosa PAO1 by Folium artemisiae argyiextractrdquo Journal of Pure and Applied Microbiology vol 7 no 1pp 261ndash270 2013

[11] J-D Yang L-B Hu W Zhou Y-F Yin J Chen and Z-QShi ldquoLysis ofMicrocystis aeruginosa with extracts from chinesemedicinal herbsrdquo International Journal of Molecular Sciencesvol 10 no 9 pp 4157ndash4167 2009

[12] A Bazire K Shioya E Soum-Soutera et al ldquoThe sigma factorAlgU plays a key role in formation of robust biofilms by

nonmucoid Pseudomonas aeruginosardquo Journal of Bacteriologyvol 192 no 12 pp 3001ndash3010 2010

[13] K D Jackson M Starkey S Kremer M R Parsek and D JWozniak ldquoIdentification of psl a locus encoding a potentialexopolysaccharide that is essential for Pseudomonas aeruginosaPAO1 biofilm formationrdquo Journal of Bacteriology vol 186 no14 pp 4466ndash4475 2004

[14] L Ma M Conover H Lu M R Parsek K Bayles and DJ Wozniak ldquoAssembly and development of the Pseudomonasaeruginosa biofilm matrixrdquo PLoS Pathogens vol 5 no 3 2009

[15] L Friedman and R Kolter ldquoGenes involved inmatrix formationin Pseudomonas aeruginosa PA14 biofilmsrdquoMolecular Microbi-ology vol 51 no 3 pp 675ndash690 2004

[16] U Remminghorst and B H A Rehm ldquoIn vitro alginatepolymerization and the functional role of Alg8 in alginateproduction by Pseudomonas aeruginosardquoApplied and Environ-mental Microbiology vol 72 no 1 pp 298ndash305 2006

[17] C Attila A Ueda and T K Wood ldquoPA2663 (PpyR) increasesbiofilm formation in Pseudomonas aeruginosa PAO1 throughthe psl operon and stimulates virulence and quorum-sensingphenotypesrdquo Applied Microbiology and Biotechnology vol 78no 2 pp 293ndash307 2008

[18] R Morgan S Kohn S-H Hwang D J Hassett and K SauerldquoBdlA a chemotaxis regulator essential for biofilmdispersion inPseudomonas aeruginosardquo Journal of Bacteriology vol 188 no21 pp 7335ndash7343 2006

[19] K Duan C Dammel J Stein H Rabin and M G SuretteldquoModulation of Pseudomonas aeruginosa gene expression byhost microflora through interspecies communicationrdquo Molec-ular Microbiology vol 50 no 5 pp 1477ndash1491 2003

[20] H Liang N Li Z DongMG Surette andKDuan ldquoTheYebCfamily protein PA0964 negatively regulates the Pseudomonasaeruginosa quinolone signal system and pyocyanin produc-tionrdquo Journal of Bacteriology vol 190 no 18 pp 6217ndash62272008

[21] G A OrsquoToole and R Kolter ldquoInitiation of biofilm formationin Pseudomonas fluorescens WCS365 proceeds via multipleconvergent signalling pathways a genetic analysisrdquo MolecularMicrobiology vol 28 no 3 pp 449ndash461 1998

[22] W Kong L Chen J Zhao et al ldquoHybrid sensor kinase PA1611in Pseudomonas aeruginosa regulates transitions between acuteand chronic infection through direct interaction with RetSrdquoMolecular Microbiology vol 88 no 4 pp 784ndash797 2013

[23] C Passariello F Berlutti L SelanM CThaller and R Pezzi ldquoArapid staining procedure to demonstrate glycocalyx productionand bacterial biofilmsrdquoNewMicrobiology vol 17 no 3 pp 225ndash230 1994

[24] J Zhao H Jiang W Cheng et al ldquoThe role of quorumsensing system in antimicrobial induced ampC expression inPseudomonas aeruginosa biofilmrdquo Journal of BasicMicrobiologyvol 55 no 5 pp 671ndash678 2015

[25] H Liang J Duan C D Sibley M G Surette and K DuanldquoIdentification ofmutants with altered phenazine production inPseudomonas aeruginosardquo Journal ofMedicalMicrobiology vol60 no 1 pp 22ndash34 2011

[26] S Yang Q Guo and K Duan ldquoPerspectives on antibacterialactivities of traditional Chinese medicine and its potentialmechanismsrdquo Chinese Journal of Biochemical and Pharmaceu-tics vol 36 no 4 pp 15ndash20 2016

[27] M Otto ldquoQuorum-sensing control in Staphylococcimdasha targetfor antimicrobial drug therapyrdquo FEMS Microbiology Lettersvol 241 no 2 pp 135ndash141 2004


[28] A R Hauser J Mecsas and D T Moir ldquoBeyond antibioticsnew therapeutic approaches for bacterial infectionsrdquo ClinicalInfectious Diseases vol 63 no 1 pp 89ndash95 2016

[29] SWagner R Sommer S Hinsberger et al ldquoNovel strategies forthe treatment of Pseudomonas aeruginosa infectionsrdquo Journal ofMedicinal Chemistry vol 59 no 13 pp 5929ndash5969 2016

[30] D G Davies M R Parsek J P Pearson B H Iglewski J WCosterton and E P Greenberg ldquoThe involvement of cell-to-cellsignals in the development of a bacterial biofilmrdquo Science vol280 no 5361 pp 295ndash298 1998

[31] N C Caiazza J H Merritt K M Brothers and G A OrsquoTooleldquoInverse regulation of biofilm formation and swarmingmotilityby Pseudomonas aeruginosa PA14rdquo Journal of Bacteriology vol189 no 9 pp 3603ndash3612 2007

[32] Y-M Zhou L Shao J-A Li et al ldquoAn efficient and novelscreening model for assessing the bioactivity of extracts againstmultidrug-resistant pseudomonas aeruginosa using caenorhab-ditis elegansrdquo Bioscience Biotechnology and Biochemistry vol75 no 9 pp 1746ndash1751 2011

[33] S A Ochoa A Cruz-Cordova G E Rodea et al ldquoPhe-notypic characterization of multidrug-resistant Pseudomonasaeruginosa strains isolated from pediatric patients associated tobiofilm formationrdquoMicrobiological Research vol 172 pp 68ndash782015

[34] M Bala S Gupte P Aggarwal M Kaur and A ManhasldquoBiofilm producing multidrug resistant Acinetobacter speciesfrom a tertiary care hospital a therapeutic challengerdquo Interna-tional Journal of Research in Medical Sciences vol 4 no 7 pp3024ndash3026 2016

[35] K Anani Y Adjrah Y Ameyapoh et al ldquoEffects of hydroe-thanolic extracts of Balanites aegyptiaca (L) Delile (Balan-itaceae) on some resistant pathogens bacteria isolated fromwoundsrdquo Journal of Ethnopharmacology vol 164 pp 16ndash212015

[36] B P Marasini P Baral P Aryal et al ldquoEvaluation of antibacte-rial activity of some traditionally used medicinal plants againsthuman pathogenic bacteriardquo BioMed Research Internationalvol 2015 Article ID 265425 6 pages 2015

[37] G Y Zuo G C Wang Y B Zhao et al ldquoScreening of Chi-nese medicinal plants for inhibition against clinical isolates ofmethicillin-resistant Staphylococcus aureus (MRSA)rdquo Journalof Ethnopharmacology vol 120 no 2 pp 287ndash290 2008

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Bacterial strains used in this study

Strainsplasmids Description Source

E coli DH10B F-mcrA(mrr-hsdRMS-mcrBC)80dlacZ ΔM15ΔlacX74 deoRrecA1 endA1 araD139 Δ(ara leu)7697 galU galK120582-rpsl nupG Invitrogen

P aeruginosa PAO1 Wild type This lab

pMS402 Expression reporter plasmid carrying the promoterlessluxCDABE Kanr Tmpr This lab

pKD-pslM pMS402 containing pslM promoter region Kanr Tmpr This studypKD-pelA pMS402 containing pelA promoter region Kanr Tmpr This studypKD-algU pMS402 containing algU promoter region Kanr Tmpr This studypKD-ppyR pMS402 containing ppyR promoter region Kanr Tmpr This studypKD-algA pMS402 containing algA promoter region Kanr Tmpr This studypKD-bdlA pMS402 containing bdlA promoter region Kanr Tmpr This study

Table 2 Reporter genes and primer sequences used

PAnumber Gene Function Primer Sequence (51015840 rarr 31015840)

PA0762 algU RNA polymerase sigma factor pKD-algU-S GCACTCGAGAGGATGCCTGAAGACCTCpKD-algU-A GTAGGATCCGATGGCGATCCGATACAG

PA2243 pslM Succinate dehydrogenasefumarate reductase flavoprotein

pKD-pslM-S ATCCTCGAGCGGTGCGCAAGAAGACCpKD-pslM-A GTTGGATCCCGTAACGCTCGCCCAGTT

PA3064 pelA Glycoside hydrolasedeacetylase

pKD-pelA-S CGTCTCGAGCTTTCCACTTTGCCACAGpKD-pelA-A TACGGATCCTACCAGAACGCCACGCT

PA3551 algAPhosphomannose isomeraseguanosine 51015840-diphospho-D-mannosepyrophosphorylase

pKD-algA-S TGACTCGAGTGAAGGCGGACTGAGACpKD-algA-A TATGGATCCGCGACCAGTTTCATC

PA2663 ppyR psl and pyoverdine operon regulator pKD-ppyR-S GCACTCGAGCACTTCTTCTGCTACAGCpKD-ppyR-A GTAGGATCCAGCACTTGCACAGCAGAC

PA1423 bdlA Biofilm dispersion locus A pKD-bdlA-S GCACTCGAGCGTCATATTTCCGACGAApKD-bdlA-A TCAGGATCCGTAGTCTTCCGATTGCG

screened for inhibitory effect against P aeruginosa biofilmsWe found that the extract of the Chinese herb H patriniaesignificantly inhibited the expression of the genes associatedwith biofilm formation in P aeruginosa PAO1 It reducedexopolysaccharide production and biofilm formation andthen altered the structure of the mature biofilms

2 Materials and Methods

21 Bacterial Strains andCulture Conditions Bacterial strainsused in this study are listed in Table 1 All the strains werecultured in LB (Luria Bertani) broth at 37∘C with orbitalshaking at 200 rpm or on LB agar plates supplemented withantibiotic of kanamycin (Kan 50 120583gmL) or trimethoprim(Tmp 300 120583gmL) where appropriate

22 Traditional Chinese Medicine Extraction TraditionalChinese medicinal herbs were selected according to theirefficacy in treatment of infection-like symptoms in Chinesemedicine They were obtained from local pharmacy (Yikang

Pharm chain store China) The pulverized herb was firstlyimmerged in 75 ethanol or deionized water respectivelyfor 2 h and then boiled for 2 h additionally (weight to solventvolume was 1 5) The extracts were filtered by filter paperand were evaporated under vacuum at 35∘C using a rotaryevaporator (Buchi Switzerland) The concentrated extractswere then freeze-dried using a lyophilizer and stored atminus80∘CThewater extractswere redissolved in deionizedwaterand ethanol extracts were redissolved in methanol and thensterilized immediately with 022120583m Iwaki filter before use

23 Gene ExpressionAssay Key genes algU [12] pslM [13 14]pelA [15] algA [16] ppyR [17] and bdlA [18] that are knownto be involved in biofilm formation in P aeruginosa wereselected to construct luxCDABE-based promoter-reporterfusions (Table 2) The reporters were constructed as previ-ously described [19 20] and were subsequently transformedinto PAO1 Using luxCDABE-based reporters gene expres-sion in liquid cultures was measured by light luminescence(in counts per second) in a Victor3 multilabel plate reader



600at the same time











Table3Eff

ecto

fwater

andethano

licherb

extractson

biofi

lm-associatedgenese

xpression

TheE

nglishnameo

fherbs

Water

extract

TheE

nglishnameo

fherbs

Ethano

licextract

algU

pslM

pelA

algA

ppyR

bdlA

algU

pslM

pelA

algA

ppyR

bdlA

Atractylodeslancea

+15

0minus25

00

0Atractylodeslancea

00

00

00

Cortex

fraxini

0minus3

minus15

minus1

00

Cortex

fraxini

minus18


00

Cyrtom

ium

fortun

ei0

minus15

minus2

minus1

00

Cyrtom

ium

fortun

ei0

00

00

0Erodium

stephanianu

mWilld

aa

aa

aa

Erodium

stephanianu

mwilld

aa

aa

aa

Foliu

martemisiae

Argyi

minus2

0minus2

0minus15

0Foliu

martemisiae

argyi

00

minus2

0minus15

0Fructusq

uisqua

lisa

aa

aa

aFructusq

uisqua

lisa

aa

aa

aHerba

agrim

oniae

aa

aa

aa

Herba

agrim

oniae

aa

aa

aa

Herba

patriniae

minus2


0minus2

Herba

patriniae

00

00

minus15

minus1

Herba

scutellariaeb

arbatae

00

0minus1

00

Herba

scutellariaeb

arbatae

00

00

0minus15

Pomegranaterin

dminus15

minus12

0minus1

00

Pomegranaterin

dminus15

minus1

minus1minus05

00

Portulacae

herba

+10

0+15

00

Portulacae

herba

00

00

00

Radixpaeoniae

minus05minus05

00

minus1

0Ra

dixpaeoniae

00

00

00

Radixsanguisorbae

minus15

minus1minus15

minus1minus05

0Ra

dixsanguisorbae

minus16

minus15

00

minus05

0Ra

dixlithospermi

0minus12

minus2

0minus1

0Ra

dixlithospermi

00

00

00

Scrophulariae

minus2

+15

00

00

Scrophulariae

minus05

0minus1

00

0Sm

oked

plum

aa

aa

aa

Smoked

plum

aa

aa

aa

Taraxacum

mongolicum

00

00

00

Taraxacum

mongolicum

0+2

00

00

Tripterygium

minus15

0minus25

00

0Tripterygium

00

minus2

0minus25

0Noteherbalextra

ctsw

ereused

at16

mgmL

Thevalues

show

nrepresentm

axim

alinhibitio

n(fo

ldchangesb

etweenexperim

entswith

andwith

outh

erbextract)

ldquo+rdquor

epresentsind

uctio

nof

gene

expressio

nby

herb

extracts


ibition

ofbiofi

lmgene

expressio

nby

herb

extracts


oeffecto

fbiofilm

gene

expressio

nby

herb

extracts


ibition

oftheg

rowth

ofbacteriaN

umbers


tthe

timep

ointsw

here

maxim

alexpressio

nwas

reachedin

thea

bsence

ofherbalextra

ct




Growth of control

0

01

02

03

04

05

06

07

08

OD

600

nm

0

500

1000

1500

2000

2500

3000

3500Ex

pres

sion

(CPS

)

3 6 9 12 15 18 21 2350Time (h)

(a)



Growth of control

0

01

02

03

04

05

06

07

OD

600

nm

0

100

200

300

400

500

600

Expr

essio

n (C

PS)

3 6 9 12 15 18 21 2350Time (h)

(b)

3 6 9 12 15 18 21 2350Time (h)

0

500

1000

1500

2000

2500

3000

Expr

essio

n (C

PS)

0

01

02

03

04

05

06

07

08

09

OD

600

nm



Growth of control

(c)

3 6 9 12 15 18 21 2350Time (d)

0

01

02

03

04

05

06

07

08

OD

600

nm

0

50

100

150

200

250

300

350

400

450

Expr

essio

n (C

PS)


(d)



















4 Conclusions



1 3 7Time (d)


0

1

2

3

4

5

6

OD

570

nm

lowast

lowast

lowastlowast

(a)

3 days 7 days

PAO1

with

Her

ba p

atrin

iae

PAO1

with

out H

erba

pat

rinia

e

(b)




(a)


1

2

3

4

5

6

7

Dia

met

er le

ngth

(cm

)

lowastlowast

(b)


WithHerba patriniae

SwarmWithout

Herba patriniae

Mature biofilm


algU darr

pslM darr



Competing Interests


Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



600at the same time











Table3Eff

ecto

fwater

andethano

licherb

extractson

biofi

lm-associatedgenese

xpression

TheE

nglishnameo

fherbs

Water

extract

TheE

nglishnameo

fherbs

Ethano

licextract

algU

pslM

pelA

algA

ppyR

bdlA

algU

pslM

pelA

algA

ppyR

bdlA

Atractylodeslancea

+15

0minus25

00

0Atractylodeslancea

00

00

00

Cortex

fraxini

0minus3

minus15

minus1

00

Cortex

fraxini

minus18


00

Cyrtom

ium

fortun

ei0

minus15

minus2

minus1

00

Cyrtom

ium

fortun

ei0

00

00

0Erodium

stephanianu

mWilld

aa

aa

aa

Erodium

stephanianu

mwilld

aa

aa

aa

Foliu

martemisiae

Argyi

minus2

0minus2

0minus15

0Foliu

martemisiae

argyi

00

minus2

0minus15

0Fructusq

uisqua

lisa

aa

aa

aFructusq

uisqua

lisa

aa

aa

aHerba

agrim

oniae

aa

aa

aa

Herba

agrim

oniae

aa

aa

aa

Herba

patriniae

minus2


0minus2

Herba

patriniae

00

00

minus15

minus1

Herba

scutellariaeb

arbatae

00

0minus1

00

Herba

scutellariaeb

arbatae

00

00

0minus15

Pomegranaterin

dminus15

minus12

0minus1

00

Pomegranaterin

dminus15

minus1

minus1minus05

00

Portulacae

herba

+10

0+15

00

Portulacae

herba

00

00

00

Radixpaeoniae

minus05minus05

00

minus1

0Ra

dixpaeoniae

00

00

00

Radixsanguisorbae

minus15

minus1minus15

minus1minus05

0Ra

dixsanguisorbae

minus16

minus15

00

minus05

0Ra

dixlithospermi

0minus12

minus2

0minus1

0Ra

dixlithospermi

00

00

00

Scrophulariae

minus2

+15

00

00

Scrophulariae

minus05

0minus1

00

0Sm

oked

plum

aa

aa

aa

Smoked

plum

aa

aa

aa

Taraxacum

mongolicum

00

00

00

Taraxacum

mongolicum

0+2

00

00

Tripterygium

minus15

0minus25

00

0Tripterygium

00

minus2

0minus25

0Noteherbalextra

ctsw

ereused

at16

mgmL

Thevalues

show

nrepresentm

axim

alinhibitio

n(fo

ldchangesb

etweenexperim

entswith

andwith

outh

erbextract)

ldquo+rdquor

epresentsind

uctio

nof

gene

expressio

nby

herb

extracts


ibition

ofbiofi

lmgene

expressio

nby

herb

extracts


oeffecto

fbiofilm

gene

expressio

nby

herb

extracts


ibition

oftheg

rowth

ofbacteriaN

umbers


tthe

timep

ointsw

here

maxim

alexpressio

nwas

reachedin

thea

bsence

ofherbalextra

ct




Growth of control

0

01

02

03

04

05

06

07

08

OD

600

nm

0

500

1000

1500

2000

2500

3000

3500Ex

pres

sion

(CPS

)

3 6 9 12 15 18 21 2350Time (h)

(a)



Growth of control

0

01

02

03

04

05

06

07

OD

600

nm

0

100

200

300

400

500

600

Expr

essio

n (C

PS)

3 6 9 12 15 18 21 2350Time (h)

(b)

3 6 9 12 15 18 21 2350Time (h)

0

500

1000

1500

2000

2500

3000

Expr

essio

n (C

PS)

0

01

02

03

04

05

06

07

08

09

OD

600

nm



Growth of control

(c)

3 6 9 12 15 18 21 2350Time (d)

0

01

02

03

04

05

06

07

08

OD

600

nm

0

50

100

150

200

250

300

350

400

450

Expr

essio

n (C

PS)


(d)



















4 Conclusions



1 3 7Time (d)


0

1

2

3

4

5

6

OD

570

nm

lowast

lowast

lowastlowast

(a)

3 days 7 days

PAO1

with

Her

ba p

atrin

iae

PAO1

with

out H

erba

pat

rinia

e

(b)




(a)


1

2

3

4

5

6

7

Dia

met

er le

ngth

(cm

)

lowastlowast

(b)


WithHerba patriniae

SwarmWithout

Herba patriniae

Mature biofilm


algU darr

pslM darr



Competing Interests


Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table3Eff

ecto

fwater

andethano

licherb

extractson

biofi

lm-associatedgenese

xpression

TheE

nglishnameo

fherbs

Water

extract

TheE

nglishnameo

fherbs

Ethano

licextract

algU

pslM

pelA

algA

ppyR

bdlA

algU

pslM

pelA

algA

ppyR

bdlA

Atractylodeslancea

+15

0minus25

00

0Atractylodeslancea

00

00

00

Cortex

fraxini

0minus3

minus15

minus1

00

Cortex

fraxini

minus18


00

Cyrtom

ium

fortun

ei0

minus15

minus2

minus1

00

Cyrtom

ium

fortun

ei0

00

00

0Erodium

stephanianu

mWilld

aa

aa

aa

Erodium

stephanianu

mwilld

aa

aa

aa

Foliu

martemisiae

Argyi

minus2

0minus2

0minus15

0Foliu

martemisiae

argyi

00

minus2

0minus15

0Fructusq

uisqua

lisa

aa

aa

aFructusq

uisqua

lisa

aa

aa

aHerba

agrim

oniae

aa

aa

aa

Herba

agrim

oniae

aa

aa

aa

Herba

patriniae

minus2


0minus2

Herba

patriniae

00

00

minus15

minus1

Herba

scutellariaeb

arbatae

00

0minus1

00

Herba

scutellariaeb

arbatae

00

00

0minus15

Pomegranaterin

dminus15

minus12

0minus1

00

Pomegranaterin

dminus15

minus1

minus1minus05

00

Portulacae

herba

+10

0+15

00

Portulacae

herba

00

00

00

Radixpaeoniae

minus05minus05

00

minus1

0Ra

dixpaeoniae

00

00

00

Radixsanguisorbae

minus15

minus1minus15

minus1minus05

0Ra

dixsanguisorbae

minus16

minus15

00

minus05

0Ra

dixlithospermi

0minus12

minus2

0minus1

0Ra

dixlithospermi

00

00

00

Scrophulariae

minus2

+15

00

00

Scrophulariae

minus05

0minus1

00

0Sm

oked

plum

aa

aa

aa

Smoked

plum

aa

aa

aa

Taraxacum

mongolicum

00

00

00

Taraxacum

mongolicum

0+2

00

00

Tripterygium

minus15

0minus25

00

0Tripterygium

00

minus2

0minus25

0Noteherbalextra

ctsw

ereused

at16

mgmL

Thevalues

show

nrepresentm

axim

alinhibitio

n(fo

ldchangesb

etweenexperim

entswith

andwith

outh

erbextract)

ldquo+rdquor

epresentsind

uctio

nof

gene

expressio

nby

herb

extracts


ibition

ofbiofi

lmgene

expressio

nby

herb

extracts


oeffecto

fbiofilm

gene

expressio

nby

herb

extracts


ibition

oftheg

rowth

ofbacteriaN

umbers


tthe

timep

ointsw

here

maxim

alexpressio

nwas

reachedin

thea

bsence

ofherbalextra

ct




Growth of control

0

01

02

03

04

05

06

07

08

OD

600

nm

0

500

1000

1500

2000

2500

3000

3500Ex

pres

sion

(CPS

)

3 6 9 12 15 18 21 2350Time (h)

(a)



Growth of control

0

01

02

03

04

05

06

07

OD

600

nm

0

100

200

300

400

500

600

Expr

essio

n (C

PS)

3 6 9 12 15 18 21 2350Time (h)

(b)

3 6 9 12 15 18 21 2350Time (h)

0

500

1000

1500

2000

2500

3000

Expr

essio

n (C

PS)

0

01

02

03

04

05

06

07

08

09

OD

600

nm



Growth of control

(c)

3 6 9 12 15 18 21 2350Time (d)

0

01

02

03

04

05

06

07

08

OD

600

nm

0

50

100

150

200

250

300

350

400

450

Expr

essio

n (C

PS)


(d)



















4 Conclusions



1 3 7Time (d)


0

1

2

3

4

5

6

OD

570

nm

lowast

lowast

lowastlowast

(a)

3 days 7 days

PAO1

with

Her

ba p

atrin

iae

PAO1

with

out H

erba

pat

rinia

e

(b)




(a)


1

2

3

4

5

6

7

Dia

met

er le

ngth

(cm

)

lowastlowast

(b)


WithHerba patriniae

SwarmWithout

Herba patriniae

Mature biofilm


algU darr

pslM darr



Competing Interests


Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Growth of control

0

01

02

03

04

05

06

07

08

OD

600

nm

0

500

1000

1500

2000

2500

3000

3500Ex

pres

sion

(CPS

)

3 6 9 12 15 18 21 2350Time (h)

(a)



Growth of control

0

01

02

03

04

05

06

07

OD

600

nm

0

100

200

300

400

500

600

Expr

essio

n (C

PS)

3 6 9 12 15 18 21 2350Time (h)

(b)

3 6 9 12 15 18 21 2350Time (h)

0

500

1000

1500

2000

2500

3000

Expr

essio

n (C

PS)

0

01

02

03

04

05

06

07

08

09

OD

600

nm



Growth of control

(c)

3 6 9 12 15 18 21 2350Time (d)

0

01

02

03

04

05

06

07

08

OD

600

nm

0

50

100

150

200

250

300

350

400

450

Expr

essio

n (C

PS)


(d)



















4 Conclusions



1 3 7Time (d)


0

1

2

3

4

5

6

OD

570

nm

lowast

lowast

lowastlowast

(a)

3 days 7 days

PAO1

with

Her

ba p

atrin

iae

PAO1

with

out H

erba

pat

rinia

e

(b)




(a)


1

2

3

4

5

6

7

Dia

met

er le

ngth

(cm

)

lowastlowast

(b)


WithHerba patriniae

SwarmWithout

Herba patriniae

Mature biofilm


algU darr

pslM darr



Competing Interests


Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














4 Conclusions



1 3 7Time (d)


0

1

2

3

4

5

6

OD

570

nm

lowast

lowast

lowastlowast

(a)

3 days 7 days

PAO1

with

Her

ba p

atrin

iae

PAO1

with

out H

erba

pat

rinia

e

(b)




(a)


1

2

3

4

5

6

7

Dia

met

er le

ngth

(cm

)

lowastlowast

(b)


WithHerba patriniae

SwarmWithout

Herba patriniae

Mature biofilm


algU darr

pslM darr



Competing Interests


Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 3 7Time (d)


0

1

2

3

4

5

6

OD

570

nm

lowast

lowast

lowastlowast

(a)

3 days 7 days

PAO1

with

Her

ba p

atrin

iae

PAO1

with

out H

erba

pat

rinia

e

(b)




(a)


1

2

3

4

5

6

7

Dia

met

er le

ngth

(cm

)

lowastlowast

(b)


WithHerba patriniae

SwarmWithout

Herba patriniae

Mature biofilm


algU darr

pslM darr



Competing Interests


Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)


1

2

3

4

5

6

7

Dia

met

er le

ngth

(cm

)

lowastlowast

(b)


WithHerba patriniae

SwarmWithout

Herba patriniae

Mature biofilm


algU darr

pslM darr



Competing Interests


Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Competing Interests


Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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Inhibition of Pseudomonas aeruginosa Biofilm Formation by ...downloads.hindawi.com/journals/bmri/2017/9584703.pdf · ResearchArticle Inhibition of Pseudomonas aeruginosa Biofilm Formation